The fundamental objective of the proposed studies is to determine the role of membrane lipid composition and fluidity in the pathogenesis of hepatic disease. Unlike most previous investigations regarding this question, these studies will take into account the heterogeneous distribution of liver plasma membrane lipids between the canalicular and basolateral domains, as well as, between the inner and outer hemileaflets of these membrane subfractions. In addition, the presence of cholesterol rich microdomains within these membranes will be investigated. The effect of alteration of lipid content and fluidity on the basic mechanisms of membrane transport will be determined. The specific aims are 1) to characterize the defect in alanine transport by basolateral rat liver plasma membranes produced by chronic ethanol consumption and to determine if this defect is caused by the ethanol induced changes in membrane lipid composition, 2) to determine if the canalicular membranes have different effects on membrane lipid composition plays a role in regulating the secretory maximum of bile acids and the production of cholestasis by lithocholate, 3) to determine if alteration of the fluidity of the inner and outer hemileaflets of basolateral and canalicular membrane transport. To achieve these specific aims purified rat canalicualr and basolateral membranes will be isolated. Membrane cholesterol will be altered using in vitro techniques. This will allow determination of the effect of cholesterol without concern that other changes, such as the number of carriers, have occurred. Transport by canalicular and basolateral membrane vesicles will be determined using a rapid millipore filtration technique. Membrane fluidity will be assessed with fluorescence anisotropy techniques with the probes 1,6 diphenyl 1,3,5-hexatriene and 12-(9-anthroyloxy) stearate. In addition the sterol probe, dehydroergosterol, will be used to probe membrane cholesterol. The physical state of the individual hemileaflets of the membrane will be assessed with Arrhenius plots of enzymes which are located asymmetrically in the membrane. The proposed studies will help to elucidate the influence of membrane lipids and fluidity on the basic mechanisms of hepatic transport and the role of membrane lipid changes in the pathogenesis of cholestatic and ethanol induced liver disease.